The bulk of animal tissue oxygen consumption is driven by a finely-balanced system in which the rate of mitochondrial catabolism of fuels is regulated largely by the flow of electrons along the electron-transport chain. Concomitant pumping of protons outward across the mitochondrial inner membrane establishes a proton electrochemical gradient or proton motive force (Δp) which drives ATP synthesis via inward flow of protons through F1F0 ATP synthase. Thus, fuel combustion, electron transport, proton flux, and ATP turnover are intimately coupled. However, a portion of the Δp is dissipated as protons flow inward independent of ATP synthase, a phenomenon termed “proton leak” or “uncoupling.” Fuel combustion and electron transport/outward proton pumping increase in response to dissipation of Δp; thus, innate mitochondrial proton leak may account for a significant amount of daily energy expenditure (estimated at between 20–40% of tissue metabolic rate)[Brand et al, Biochim. Biophys. Acta, 1187:132–139 (1994); Rolfe et al., Am. J. Physiol., 276:C692–C699 (1999)].
The first indication that specific proteins may underlie mammalian mitochondrial proton leak emerged from studies of brown adipose tissue (BAT), a specialized tissue in which a large proportion of mitochondrial oxygen consumption is uncoupled from ATP synthesis under conditions in which adaptational thermogenesis is triggered (i.e. cold-exposure in rodents)[Nicholls and Locke, Physiol. Rev., 64:1–64 (1984)]. The heat-generating futile cycling of the BAT mitochondrial proton circuit was found to be associated with a specific protein termed uncoupling protein (UCP, subsequently named UCP1)[Nicholls and Locke, supra; Ricquier et al., FASEB J., 5:2237–2242 (1991)].
UCPs were first found and described in the brown fat cells of hibernating animals, such as bears. UCPs were believed to help such hibernators and other cold-weather adapted animals maintain core body temperatures in cold weather by raising their body's resting metabolic rate. Because humans possess relatively small quantities of brown adipose tissue, UCPs were originally thought to play a minor role in human metabolism.
Several different human uncoupling proteins have now been described. [See, generally, Gura, Science, 280:1369–1370 (1998)]. The human uncoupling protein referred to as UCP1 was identified by Nicholls et al. Nicholls et al. showed that the inner membrane of brown fat cell mitochondria was very permeable to proteins, and the investigators traced the observed permeability to a protein, called UCP1, in the mitochondrial membrane. Nicholls et al. reported that the UCP1, by creating such permeability, reduced the number of ATPs that can be made from a food source, thus raising body metabolic rate and generating heat. [Nicholls et al., Physiol. Rev., 64, 1–64 (1984)].
It was later found that UCP1 is indeed expressed only in brown adipose tissue [Bouillaud et al., Proc. Natl. Acad. Sci. 82:445–448 (1985); Jacobsson et al., J. Biol. Chem., 260:16250–16254 (1985)]. Genetic mapping studies have shown that the human UCP1 gene is located on chromosome 4. [Cassard et al., J. Cell. Biochem., 43:255–264 (1990)].
Despite confinement of UCP1 to BAT under most conditions, significant proton leak occurs in all tissues in which it has been measured, leading to the possibility that UCPs are present body-wide and impact whole-animal metabolic rate. To date, four putative UCP homologs have been identified, with homolog-specific tissue expression patterns [see Adams, J. Nutr., 130:711–714 (2000)].
Another human UCP, referred to as UCPH or UCP2, has also been described. [Gimeno et al., Diabetes, 46:900–906 (1997); Fleury et al., Nat. Genet., 15:269–272 (1997); Boss et al., FEBS Letters, 408:39–42 (1997); see also, Wolf, Nutr. Rev., 55:178–179 (1997)]. Fleury et al. teach that the UCP2 protein has 59% amino acid identity to UCP1, and that UCP2 maps to regions of human chromosome 11 which have been linked to hyperinsulinaemia and obesity. [Fleury et al., supra]. It has also been reported that UCP2 is expressed in a variety of adult tissues, such as brain and muscle and fat cells. [Gimeno et al., supra, and Fleury et al., supra].
A third human UCP, UCP3, was recently described in Boss et al., supra; Vidal-Puig et al., Biochem. Biophys. Res. Comm., 235:79–82 (1997); Solanes et al., J. Biol. Chem., 272:25433–25436 (1997); and Gong et al., J. Biol. Chem., 272:24129–24132 (1997). [See also Great Britain Patent No. 9716886]. Solanes et al. report that unlike UCP1 and UCP2, UCP3 is expressed preferentially in human skeletal muscle, and that the UCP3 gene maps to human chromosome 11, adjacent to the UCP2 gene. [Solanes et al., supra]. Gong et al. describe that the UCP3 expression can be regulated by known thermogenic stimuli, such as thyroid hormone, beta3-andrenergic agonists and leptin. [Gong et al., supra].
To characterize a putative UCP, an in vitro assay measuring mitochondrial membrane potential (ΔΨm) may be used. Ectopic expression of UCP homologs in mammalian cell lines and yeast leads to a drop in ΔΨm, consistent with uncoupling under these conditions. As a negative control in such experiments, it is preferable to use a molecule that is a mitochondria-localized carrier that exchanges compounds in an electroneutral manner. Human 2-oxoglutarate (human OGC) exhibits these characteristics. Furthermore, it was reported that expression of human OGC in transformed yeast did not affect mitochondrial function [Sanchis et al., J. Biol. Chem. 273(51):34611–34615 (1998)].